The overarching goal of our research is to develop new biomarkers of islet autoimmunity and to translate these discoveries to prevention of human Type 1A diabetes (T1D). During the last funding period, we have developed a new generation of assays to detect autoantibodies to islet and non-islet autoantigens with higher sensitivity and predictive value. The proposed studies will widen the spectrum of relevant autoantigens and diseases as well as provide opportunities to reduce cost and expand applications. While our recent findings have strengthened the evidence for the primary role of T-cells autoreactive to (pro)insulin in advanced human islet autoimmunity, we still lack robust T-cell biomarkers that could clearly reflect activity of autoreactive T cells, especially at the earliest stages of islet autoimmunity or during immunomodulation to prevent progression to diabetes. Improved assays for islet autoantibodies and autoreactive T-cells, and better understanding of their relationship and potential genetic determinants, will enhance our understanding of the pathogenesis of T1D and improve prediction of progression from subclinical to clinical disease. Specific Aims of this project include: 1.Multiplex and optimize novel non-radioactive assays to a wide spectrum of islet and non-islet autoantigens. A. Develop a novel system for simultaneous measurement of IAA, GADA, IA-2A and ZnT8A and validate the multiplex assay in a large population of patients with T1D and LADA. B. Add non-islet autoantigens to the optimal multiplex islet autoantibody assay to develop a robust screening tool for multiple autoimmune phenotypes 2. Develop molecular based, non-cellular T cell biomarkers that do not requiring living T cells and can be assessed using whole blood samples. A. Determine common V-genes preferentially used by T1D- associated TCR sequences. B. Determine public TCR sequences in individuals with anti-islet autoimmunity and clinical T1D by deep sequencing specific V-genes 3. Explore sequential development of the pre-T1D autoimmune phenotype including multiple iAbs and public TCR sequences (identified in Specific Aim 2.B) in 90 children prospectively followed from birth for development of islet autoimmunity and T1D and in 90 HLA-DR-DQ -matched controls.